Methods and compositions comprising ursolic acid and/or resveratrol for treating diabetes, or cancer

ABSTRACT

Certain embodiments are directed to methods and compositions for treating obesity, diabetes, and/or cancer with a combination of ursolic acid and resveratrol.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/918,378 filed Jul. 1, 2020; which is a continuation of U.S.application Ser. No. 16/813,017 filed Mar. 9, 2020, which is acontinuation of application Ser. No. 16/221,094 filed Dec. 14, 2018 (nowU.S. Pat. No. 10,583,145), which is a continuation of application Ser.No. 15/308,427 (now U.S. Pat. No. 10,155,003) filed Nov. 2, 2016, whichis a national phase application under 35 USC § 371 of InternationalApplication No. PCT/US2015/029224 filed May 5, 2015, which claimspriority to U.S. Provisional Application No. 61/988,859 filed May 5,2014, each of which is hereby incorporated in their entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under CA54174 awarded bythe National Institutes of Health. The government has certain rights inthe invention.

BACKGROUND

The recent explosion of obesity, type II diabetes, heart disease, andsome cancers in the United States can be largely attributed to two keywell-known factors, a lack of a healthy diet and reduced physicalactivity. Type II diabetes, high blood pressure, and heart disease arecomponents of the “metabolic syndrome”, an umbrella term for maladiestypically developed as a consequence of being overweight/obese andphysically inactive. Studies have shown that physical activity andreduced calorie intake, or “calorie restriction” inhibit degenerativeand chronic disease, especially those of the metabolic syndrome andcancer.

Obesity is a chronic disease that is prevalent in modern society and isassociated not only with a social stigma, but also with decreased lifespan and numerous medical problems—obesity is a primary risk factor fortype II diabetes mellitus and is a strong risk factor for cardiovasculardisease and cancer as well. Much of this obesity-induced pathology canbe attributed to the strong association with dyslipidemia, hypertension,and insulin resistance. Many studies have demonstrated that reduction inobesity by diet and exercise reduces these risk factors dramatically.Unfortunately these treatments are largely unsuccessful with a failurerate reaching 95%. This failure may be due to the fact that thecondition is strongly associated with genetically inherited factors thatcontribute to increased appetite, preference for highly caloric foods,reduced physical activity, and increased lipogenic metabolism. Thisindicates that people inheriting these genetic traits are prone tobecoming obese regardless of their efforts to combat the condition.

Existing therapies for obesity include standard diets and exercise, verylow calorie diets, behavioral therapy, pharmacotherapy involvingappetite suppressants, thermogenic drugs, and food absorptioninhibitors, mechanical devices such as jaw wiring, waist cords,balloons, and surgery (Jung and Chong. Clinical Endocrinology. 1991,35:11-20; Bray. Am J Clin Nutr. 1992, 55:538S-544S). Because dieting andexercise produce only modest results, researchers have searched for acompound to accelerate the loss of body fat. However, of the drugscurrently allowed for use over a long time period, sibutramine, anappetite depressant, and orlistat, a lipase inhibitor, have side effectsincluding headaches, polydipsia (serious thirst), insomnia,constipation, hypertension, and increased pulse rate and fecalincontinence, frequent or urgent bowel movements, steatorrhea, and areduction in the absorption of fat-soluble vitamins, respectively.

There remains a need for additional compositions and methods for thetreatment of obesity, diabetes, and various cancers.

SUMMARY

Certain embodiments are directed to methods and compositions fortreating obesity, diabetes, and/or cancer with a combination of ursolicacid and resveratrol. Certain embodiments are directed to compositionsfor supplementing a human diet. Ursolic acid is a phytonutrient found ina wide variety of food products and herbs especially apples, olives, androsemary. As described herein, studies indicate that ursolic acidadministration can mimic and enhance the effects of calorie restrictionand exercise. Ursolic acid inhibits chronic inflammation and increasesthe effectiveness of anti-diabetic drugs such as Rosiglitazone andMetformin on glucose uptake into insulin resistant fat cells.Resveratrol is a phytonutrient found in the skin of red grapes and inother fruits. Resveratrol exerts many of its beneficial effects throughits ability to mimic and enhance the effects of calorie restriction andexercise.

Certain embodiments are directed to methods for treating obesitycomprising administering an effective amount of ursolic acid and aneffective amount of resveratrol to an obese subject. In certain aspectsursolic acid is administered at a dose of between 50, 100, 150, 200,250, 300 to 250, 300, 350, 400, 450, 500, 550, 600 mg/day, including allvalues and ranges there between. In certain aspects 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, or 600 mg of ursolic acid isadministered. In a further aspect the dose of ursolic acid isadministered in one dose or in multiple doses over 0.5, 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 minutes, hours or days. In certain aspects resveratrolis administered at a dose of between 50, 100, 150, 200, 250, 300 to 250,300, 350, 400, 450, 500, 550, 600 mg/day, including all values andranges there between. In certain aspects 50, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, or 600 mg of resveratrol is administered. In afurther aspect the dose of resveratrol is administered in one dose or inmultiple doses over 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, hoursor days. In certain aspects ursolic acid and resveratrol areadministered at a ratio of 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, or 1:4,including all values and ranges there between. In certain aspectsursolic acid and resveratrol are administered individually. Individualadministration refers to the compounds being formulated is separateformulations. The compounds when administered individually can beadministered at the same time or within about 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 minutes, hours, or days. In certain aspects ursolic acid andresveratrol are formulated in the same composition. Ursolic acid and/orresveratrol can be formulated as a tablet, a capsule, a concentrate, apowder, a beverage, a baked good, chocolate, caramel, cookie, bar,and/or snack. In certain aspects ursolic acid and/or resveratrol areadministered orally.

In certain aspects administration of ursolic acid is prior toadministration of resveratrol. In a further aspect administration ofursolic acid is concurrent with resveratrol. In still a further aspectsadministration of resveratrol is prior to administration of ursolicacid.

In certain aspects ursolic acid and resveratrol are administered to asubject having a body mass index (BMI) of 25, 30, 40, 50, or greater.The subject can be diagnosed with pre-diabetes.

Certain embodiments are directed to methods and compositions fortreating diabetes comprising administering an effective amount ofursolic acid in combination with an effective amount of resveratrol tothe subject. In certain aspects ursolic acid is administered at a doseof between 50, 100, 150, 200, 250, 300 to 250, 300, 350, 400, 450, 500,550, 600 mg/day, including all values and ranges there between. Incertain aspects 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or600 mg of ursolic acid is administered. In a further aspect the dose ofursolic acid is administered in one dose or in multiple doses over 0.5,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, hours or days. In certainaspects resveratrol is administered at a dose of between 50, 100, 150,200, 250, 300 to 250, 300, 350, 400, 450, 500, 550, 600 mg/day,including all values and ranges there between. In certain aspects 50,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 mg ofresveratrol is administered. In a further aspect the dose of resveratrolis administered in one dose or in multiple doses over 0.5, 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 minutes, hours or days. In certain aspects ursolicacid and resveratrol are administered at a ratio of 4:1, 3:1, 2:1, 1:1,1:2, 1:3, or 1:4, including all values and ranges there between. Incertain aspects ursolic acid and resveratrol are administeredindividually. Individual administration refers to the compounds beingformulated is separate formulations. The compounds when administeredindividually can be administered at the same time or within about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 minutes, hours, or days. In certain aspectsursolic acid and resveratrol are formulated in the same composition.Ursolic acid and/or resveratrol can be formulated as a tablet, acapsule, a concentrate, a powder, a beverage, a baked good, chocolate,caramel, cookie, bar, and/or snack. In certain aspects ursolic acidand/or resveratrol are administered orally.

In certain aspects administration of ursolic acid is prior toadministration of resveratrol. In a further aspect administration ofursolic acid is concurrent with resveratrol. In still a further aspectsadministration of resveratrol is prior to administration of ursolicacid.

Certain embodiments are directed to methods and compositions fortreating pre-diabetes comprising administering an effective amount ofursolic acid in combination with an effective amount of resveratrol to asubject. In certain aspects ursolic acid is administered at a dose ofbetween 50, 100, 150, 200, 250, 300 to 250, 300, 350, 400, 450, 500,550, 600 mg/day, including all values and ranges there between. Incertain aspects 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or600 mg of ursolic acid is administered. In a further aspect the dose ofursolic acid is administered in one dose or in multiple doses over 0.5,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, hours or days. In certainaspects resveratrol is administered at a dose of between 50, 100, 150,200, 250, 300 to 250, 300, 350, 400, 450, 500, 550, 600 mg/day,including all values and ranges there between. In certain aspects 50,100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 mg ofresveratrol is administered. In a further aspect the dose of resveratrolis administered in one dose or in multiple doses over 0.5, 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 minutes, hours or days. In certain aspects ursolicacid and resveratrol are administered at a ratio of 4:1, 3:1, 2:1, 1:1,1:2, 1:3, or 1:4, including all values and ranges there between. Incertain aspects ursolic acid and resveratrol are administeredindividually. Individual administration refers to the compounds beingformulated is separate formulations. The compounds when administeredindividually can be administered at the same time or within about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 minutes, hours, or days. In certain aspectsursolic acid and resveratrol are formulated in the same composition.Ursolic acid and/or resveratrol can be formulated as a tablet, acapsule, a concentrate, a powder, a beverage, a baked good, chocolate,caramel, cookie, bar, and/or snack. In certain aspects ursolic acidand/or resveratrol are administered orally.

In certain aspects administration of ursolic acid is prior toadministration of resveratrol. In a further aspect administration ofursolic acid is concurrent with resveratrol. In still a further aspectsadministration of resveratrol is prior to administration of ursolicacid.

Still further embodiments are directed to compositions and methods fortreating skin cancer comprising administering an effective amount ofursolic acid in combination with an effective amount of resveratrol. Incertain aspects ursolic acid is administered at a dose of between 50,100, 150, 200, 250, 300 to 250, 300, 350, 400, 450, 500, 550, 600mg/day, including all values and ranges there between. In certainaspects 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 mgof ursolic acid is administered. In a further aspect the dose of ursolicacid is administered in one dose or in multiple doses over 0.5, 1, 2, 3,4, 5, 6, 7, 8, 9, or 10 minutes, hours or days. In certain aspectsresveratrol is administered at a dose of between 50, 100, 150, 200, 250,300 to 250, 300, 350, 400, 450, 500, 550, 600 mg/day, including allvalues and ranges there between. In certain aspects 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, or 600 mg of resveratrol isadministered. In a further aspect the dose of resveratrol isadministered in one dose or in multiple doses over 0.5, 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 minutes, hours or days. In certain aspects ursolicacid and resveratrol are administered at a ratio of 4:1, 3:1, 2:1, 1:1,1:2, 1:3, or 1:4, including all values and ranges there between. Incertain aspects ursolic acid and resveratrol are administeredindividually. Individual administration refers to the compounds beingformulated is separate formulations. The compounds when administeredindividually can be administered at the same time or within about 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 minutes, hours, or days. In certain aspectsursolic acid and resveratrol are formulated in the same composition.Ursolic acid and/or resveratrol can be formulated as a topical solution(crème, ointment, gel, etc.), tablet, a capsule, a concentrate, apowder, a beverage, a baked good, chocolate, caramel, cookie, bar,and/or snack. In certain aspects ursolic acid and/or resveratrol areadministered topically. In certain aspects ursolic acid and/orresveratrol are administered orally.

In certain aspects administration of ursolic acid is prior toadministration of resveratrol. In a further aspect administration ofursolic acid is concurrent with resveratrol. In still a further aspectsadministration of resveratrol is prior to administration of ursolicacid.

The term “isolated” can refer to a compound that is substantially freeof cellular material, bacterial material, viral material, culture medium(when produced by recombinant DNA techniques), chemical precursors orother chemicals (when chemically synthesized). Moreover, an isolatedcompound refers to one that can be administered to a subject as anisolated compound; in other words, the compound may not simply beconsidered “isolated” if it is adhered to a column or embedded in anagarose gel.

Other embodiments of the invention are discussed throughout thisapplication. Any embodiment discussed with respect to one aspect of theinvention applies to other aspects of the invention as well and viceversa. Each embodiment described herein is understood to be anembodiment of the invention that is applicable to other aspects of theinvention. It is contemplated that any embodiment discussed herein canbe implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions and kits of theinvention can be used to achieve methods of the invention.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofthe specification embodiments presented herein.

FIG. 1 . Ursolic acid (UA) and resveratrol (RES) synergisticallyincrease cellular glucose uptake.

FIG. 2 . RES and UA synergistically activate AMPK in a mouse model forskin cancer.

FIGS. 3A-B. RES sensitizes MT1/2 mouse papilloma cells and Ca3/7 mousesquamous carcinoma cells to UA. (FIG. 3A) MT1/2 cells co-treated with UAand RES had lower IC₅₀ values relative to treatment with UA alone. (FIG.3B) Ca3/7 cells co-treated with UA and RES had lower IC₅₀ valuesrelative to treatment with UA alone. (For A and B, n=3 and * indicatesp<0.05 between UA+veh and UA+50 μM RES, $ indicates p<0.05 betweenUA+veh and UA+100 μM RES).

FIG. 4 . A higher concentration of RES increases the sensitivity ofCa3/7 cells to UA (n=3, * indicates p<0.05).

FIG. 5 . UA and RES synergistically inhibit viability of human lungadenocarcinoma cell line A549 (n=3, * indicates p<0.05 between UA+vehand UA+10 μM RES, $ indicates p<0.05 between UA+veh and UA+25 μM RES).

DESCRIPTION

Certain embodiments are directed to treating obesity, diabetes, andcancer with a combination of ursolic acid and resveratrol. Ursolic acidinhibits chronic inflammation. In diabetic rats, dietary ursolic aciddecreases resting glucose levels, and improves glucose tolerance andinsulin sensitivity. Also, in a rat model of hypertension, ursolic aciddecreases blood pressure, resting glucose, LDL cholesterol, andtriglycerides to near control levels. Furthermore, mice fed a high-fatdiet containing ursolic acid showed a decrease in body weight,normalized glucose levels, an elevated level of circulating insulin, anda dramatic decrease in liver lipids compared to the control mice whichbecame obese and diabetic. These results suggest that ursolic acid hasthe potential to reduce the risk of obesity and diabetes. In additionursolic acid increases the effectiveness of anti-diabetic drugs such asRosiglitazone and Metformin on glucose uptake into insulin resistant fatcells. Ursolic acid is able to reduce fat accumulation and increasemuscle mass gain when in a fed state and to induce fat burning andpreserve muscle mass when in a fasted stated. In experimental animalsursolic acid also is effective in preventing the induction of skin,breast, prostate, and colon cancer throughout its anti-inflammatoryeffect.

Resveratrol is a phytonutrient found in the skin of red grapes and inother fruits. Like ursolic acid, it also exerts many of its beneficialeffects through its ability to mimic calorie restriction and exercise.The effects of resveratrol are currently a topic of numerous animal andhuman studies. In experimental animal studies, resveratrol has beenshown to have anti-inflammatory, blood sugar lowering, and otherbeneficial cardiovascular effects. Resveratrol inhibited weight gain inrats fed atherogenic diet (1% cholesterol) with an accompanying decreasein liver inflammation. With regards to the metabolic syndrome,resveratrol improves insulin sensitivity in mice fed a high caloriediet. In type II diabetic humans, insulin sensitivity was improved bythe fourth week following daily resveratrol intake. However, whileseveral human studies have reported generally positive effects,resveratrol may have lesser benefits except at very high doses. Likeursolic acid, resveratrol appears to enhance the activity ofanti-diabetic drugs. In addition, resveratrol has been shown to inhibitthe induction of skin and breast cancer.

Animal studies using either ursolic acid or resveratrol show the samepositive effects—preventing obesity, diabetes, and cancer. Recently theinventors have found that a combination of both ursolic acid andresveratrol has very potent synergistic effects on several criticaldiagnostic markers related to obesity, type II diabetes, and cancer.Recent results show that ursolic acid and resveratrol interactsynergistically to dramatically and significantly increase theenergy-sensing pathways (AMPK) and decrease the MTOR pathway. Bothcalorie restriction and exercises have strong effects on these pathways.In addition, a combination of ursolic acid and resveratrol strongly andsignificantly decreases the inflammatory pathway (NFKB) in the liver andpancreas induced by high fat diets. Both ursolic acid and resveratrolalso synergistically increase glucose transport into muscle cells andincrease adiponectin secretion from fat cells in diabetic experimentalanimals. Both of these activities counteract insulin resistance,obesity, and diabetes. The inventors have also found that ursolic acidand resveratrol can synergistically inhibit the inflammatory pathway inhuman monocytes treated to increase the activity of this pathway. Also,a combination of ursolic acid and resveratrol has an even greater effectwhen given with a partial calorie restriction and moderate exercise.

I. PRE-DIABETES SYNDROME

Pre-diabetes is the state in which some but not all of the diagnosticcriteria for diabetes are met, including impaired fasting glycemia orimpaired fasting glucose (IFG) and impaired glucose tolerance (IGT). IFGrefers to a condition in which the fasting blood glucose is elevatedabove what is considered normal levels but is not high enough to beclassified as diabetes mellitus. Fasting blood glucose levels are in acontinuum within a given population, with higher fasting glucose levelscorresponding to a higher risk for complications caused by the highglucose levels. IFG is defined as a fasting glucose that is higher thanthe upper limit of normal, but not high enough to be classified asdiabetes mellitus. Some patients with impaired fasting glucose can alsobe diagnosed with IGT as described below, but many have normal responsesto a glucose tolerance test.

IFG is considered a pre-diabetic state, associated with insulinresistance, increased mortality, and increased risk of cardiovascularpathology, although of lesser risk than IGT (Barr et al. Circulation.2007, 116(2):151-157). There is a 50% risk over 10 years of progressingto overt diabetes, but many newly identified IFG patients progress todiabetes in less than three years (Nichols et al. Diabetes Care. 2007,30(2):228-233).

IGT is a pre-diabetic state of dysglycemia, that is associated withinsulin resistance and increased risk of cardiovascular pathology. IGTmay precede type 2 diabetes mellitus by many years. IGT is also a riskfactor for mortality (Nichols et al. Diabetes Care. 2007,30(2):228-233).

Following the ADA criteria, pre-diabetes can be diagnosed with a bloodtest with any of the following results: (1) Fasting blood sugar(glucose) level from 100-125 mg/dL (5.6-6.9 mM); (2) A blood sugar levelof 140 to 199 mg/dL (7.8 to 11.0 mM) two hours after ingesting thestandardized 75 gram glucose solution in the glucose tolerance test; (3)Glycated hemoglobin between 5.7 and 6.4%.

Diabetes can be diagnosed with a blood test with any of the followingresults: (1) Fasting blood sugar (glucose) level ≥126 mg/dL (7.0 mM);(2) A blood sugar level ≥200 mg/dL (11.1 mM) two hours after ingestingthe standardized 75 gram glucose solution in the glucose tolerance test;(3) Glycated hemoglobin ≥6.5%; (4) Symptoms of hyperglycemia and casualplasma glucose ≥200 mg/dL (11.1 mM).

II. OBESITY

Obesity refers to a condition where excessive fat accumulates within thebody. In general, when a person's body mass index (BMI) is greater than30, they are diagnosed as obese. Body mass index (BMI) is a widely usedmethod for estimating body fat mass and is an accurate reflection ofbody fat percentage in the majority of the adult population. BMI iscalculated by dividing the subject's mass by the square of his or herheight, typically expressed either in metric or US “Customary” units askg/m′ or pounds×703/inches. A person with a BMI of 30.0 or greater isdefined as obese, with higher BMI values being further classified assevere obesity (35.0 to 40), morbid obesity (40.0 to 45), and superobese (BMI ≥45).

Obesity is caused by an energy imbalance over a long period when anexcessive amount of calories are ingested with respect to the amount ofenergy being expended. Treatment of obesity normally requires behaviortherapy as well as a reduction of calories ingested and/or an increasein the amount of calories expended.

Low levels of the protein adiponectin have been associated with a higherrisk of developing metabolic syndromes such as obesity. In certainaspects the methods described herein result in the upregulation ofadiponectin. Adiponectin (also referred to as GBP-28, apM1, AdipoQ andAcrp30) is a 244 amino acid protein that in humans is encoded by theADIPOQ gene. It is a protein hormone that modulates a number ofmetabolic processes, including glucose regulation and fatty acidoxidation. Adiponectin is secreted into the bloodstream from adiposetissue and also from the placenta in pregnancy (Chen et al.Diabetalogica. 2006, 49(6):1292-1302). In the bloodstream, adiponectinaccounts for approximately 0.01% of all plasma protein at around 5-10μg/mL and is very abundant in plasma relative to many hormones. Levelsof the hormone are inversely correlated with body fat percentage inadults, while the association in infants and young children is lessclear (Ukkola and Santaniemi. J Mol Med. 2002, 80(11):696-702).

Transgenic mice with increased adiponectin show impaired adipocytedifferentiation and increased energy expenditure associated with proteinuncoupling (Bauche et al. Endocrinology. 148(4):1539-1549). The hormoneplays a role in the suppression of the metabolic derangements that mayresult in type 2 diabetes, obesity, atherosclerosis, non-alcoholic fattyliver disease (NAFLD) and an independent risk factor for metabolicsyndrome (Diez and Iglesias. Eur J Endocrinol. 2003, 148(3):293-300;Ukkola and Santaniemi. J Mol Med. 2002, 80(11):696-702; Renaldi et al.Acta Med Indones. 2009, 41(1):20-24). Adiponectin in combination withleptin has been shown to completely reverse insulin resistance in mice(Yamauchi et al. Nat Med. 2001, 7(8):941-946). Levels of adiponectin arereduced in diabetics compared to non-diabetics. Weight reductionsignificantly increases circulating levels (Coppola et al, Int JCardiol. 2008 134(3):414-416).

III. SKIN CANCER

There are over one million new cases of skin cancer diagnosed in theUnited States each year (Rogers et al. Arch. Dermatol. 2010,146(3):283-287). In addition, skin cancer is associated with a 15-30%increased risk of other forms of cancer (Kahn et al. JAMA. 1998280(10):910-912; Krueger et al. Can. J Public Health. 2010,101(4):123-27). This indicates the importance of mechanisms to preventor treat skin cancer. Skin cancer consists primarily of melanoma, basalcell carcinoma, and squamous cell carcinoma. Skin cancers have higherrisk with increased UV exposure (Boscoe and Schymura. BMC Cancer. 20066:264; Lea et al. Ann. Epidemiol. 2007, 17(6):447-453) and higherprevalence in individuals with inflammatory disorders (Frentz and Olsen.Br. J Dermatol. 1999, 140(2):237-242; Long et al. Clin. Gastroenterol.Hepatol. 2010, 8(3):268-274).

Cancer develops via three phases: initiation, promotion, andprogression. During initiation, DNA mutations lead to activation ofoncogenes and inactivation of tumor suppressor genes. DNA mutations inskin cancer are caused by environmental insults such as UV exposure andpolycyclic aromatic hydrocarbons (PAHs). PAHs are present in smoke andcan form DNA adducts when metabolized (Baird et al Eniron. Mol. Mutagen.2005, 45(2-3):106-144; Boffetta et al, Cancer Causes Control. 19978(3):444-472). In tumor promotion, activated oncogenes and inactivatedtumor suppressor genes cause constitutive activation of tumor-promotingsignal transduction pathways. These pathways increase factors for cellproliferation, cell growth, resistance to apoptosis, and angiogenesis(Walaszek et al. Chest. 2004, 125(5 Suppl):128S-133 S). Finally, tumorprogression occurs when additional genetic alterations allow tumor cellsto enter the bloodstream and metastasize to distant organ sites (Gialeliet al. FEBS J. 2011, 278(1): 16-27).

The tumor promotion phase is characterized by abnormal activity of manysignal transduction pathways, including the nuclear factor kappa B(NFKB) pathway. NFKB is a transcription factor that typically exists asheterodimer of p65 and p50 subunits. A number of tumor-promoting factorsincluding UV light (Kato et al. Mol Cell 2003, 12(4):829-839; Lee et al.Int J Mot Med 2009, 23(5):679-684) and various tobacco constituents(Rajendrasozhan et al. Pulm Pharmacol Ther 2010, 23(3):172-181;Tsurutani et al. Carcinogenesis 2005, 26(7):1182-1195) activate the NFKBpathway. These stimuli phosphorylate and activate kinases thatphosphorylate the NFKB inhibitor 1 KBa, targeting it for degradation byproteaseomes. Stimuli including UV light (Laszlo and Wu. PhotochemPhotobiol 2008, 84(6):1564-1568), smoke (Rajendrasozhan et al. PulmPharmacol Ther 2010, 23 (3): 172-181), or inflammatory agents (Hsing etal. PLoS One 2011, 6(3):e17598) also phosphorylate the active p65subunit of NFKB at serines 536 and/or 276, allowing it to recruithistone acetyltransferases (HATs) (Chen et al. Mol Cell Biol 2005,25(18):7966-7975). These HATs acetylate p65 at a number of lysineresidues, resulting in dissociation of NFKB from its inhibitor IKBa(Chen et al. EMBO J 2002, 21(23):6539-6548; Kiernan et al. J Blot Chem2003, 278(4):2758-2766). Once free from IKBa, NFKB can translocate tothe nucleus where it transcribes factors for cell proliferation,inflammation, resistance to apoptosis, angiogenesis, and metastasis(Kundu and Surh. Mutat Res 2008, 659(1-2):15-30).

Certain phytochemicals present in fruits and vegetables can inhibitcancer formation and growth in many experimental models. Also,epidemiological studies show that consumption of phytochemical-richfruits and vegetables decreases the risk of many cancer types includingskin cancer (Ibiebele et al. Am J Clin Nutr 2007, 85(5):1401-1408; Kuneet al. Nutr Cancer 1992, 18(3):237-244). Resveratrol inhibits formationof many tumor types in animal models, including those of the skin (Azizet al. FASEB J 2005, 19(9):1193-1195; Kapadia et al. Pharmacol Res 2002,45(6):499-505). Ursolic acid (UA) also inhibits tumor formation in anumber of models including chemically-induced skin cancer (Huang et al.Cancer Res 1994, 54(3):701-708; Tokuda et al. Cancer Lett 1986,33(3):279-285). Resveratrol and UA also inhibit NFKB signaling.

In addition to their anti-cancer effects, a wide range of phytochemicalsand plant extracts have also been shown to inhibit the metabolicsyndrome, including insulin resistance and diabetes (Xia and Weng. JDiabetes 2010, 2(4):243-249; Graf et al. Curr Opin Investig Drugs 2010,11(10):1107-1115; Cherniack. Nutrition 2011, 27(6):617-623; Leiherer etal. Vascul Pharmacol 2013, 58(1-2):3-20). In many cases these effectsare associated with AMP-activated kinase (AMPK), which also mediates theactivities of prescribed anti-diabetic drugs like metformin (Hattori etal. Hypertension 2006, 47(6):1183-1188; Musi et al. Diabetes 2002,51(7):2074-2081; Zhou et al. J Clin Invest 2001, 108(8):1167-1174;Hardie et al. Chem Biol 2012, 19(10):1222-1236). AMPK is activated byexercise in humans and animals as indicated by an increase inphosphorylation of threonine 172 (Birk and Wojtaszewski. J Physiol 2006,577(Pt 3):1021-1032; Hoene et al. J Physiol 2009, 587(Pt 1):241-252;Koopman et al. Am J Physiol Endocrinol Metab 2006, 290(6):E1245-1252).AMPK also plays a key role in NFKB inhibition by different compoundsused to treat diabetes (Hattori et al. Hypertension 2006,47(6):1183-1188; Tomizawa et al. Metabolism 2011, 60(4):513-522).

Resveratrol and UA improve symptoms of metabolic syndromes and diabeticsymptoms in both animal and humans (Baur et al. Nature 2006,444(7117):337-342; Brasnyo et al. Br J Nutr 2011, 106(3):383-389; Janget al. Int Immunopharmacol 2009, 9(1):113-119; Somova et al.Phytomedicine 2003, 10(2-3):115-121). Resveratrol activates AMPK in manyorganisms including humans (Timmers et al. Cell Metab 2011,14(5):612-622; Xu and Si. Nutr Res 2012, 32(9):648-658), and the fulleffects of resveratrol on the metabolic syndrome depend on AMPK activity(Um et al. Diabetes 2010, 59(3):554-563). The cytotoxic effects of UA indifferent cancer cell lines are also dependent on AMPK activation (Sonet al. Phytother Res 2013; Zheng et al. Biochem Biophys Res Commun 2012,419(4):741-747). Finally, AMPK activity suppresses tumorigenesis intumor-susceptible animals (Huang et al. Biochem J 2008, 412(2):211-221;Faubert et al. Cell Metab 2013, 17(1):113-124). These results indicatethe anti-cancer and anti-diabetic effects of many natural compounds likeresveratrol and UA may be mediated by AMPK activation.

There are many potential synergistic mechanisms for differentphytochemical combinations. One drug may modulate the metabolism of theother (Kimura et al. Food Chem Toxicol 2010, 48(1):429-435; Taesotikulet al. Drug Metab Pharmacokinet 2011, 26(2):154-161), or impact itsability to enter the bloodstream (Lu et al. J Nutr Biochem 2005,16(1):23-30) or the cell (Suganuma et al. Cancer Res 1999, 59(1):44-47).Drugs can also enhance the ability of each other to induce similardownstream effects, many times by acting along different points on cellregulatory systems (Khafif et al. Carcinogenesis 1998, 19(3):419-424;Saw et al. Biopharm Drug Dispos 2011, 32(5):289-300).

Resveratrol has been shown to synergistically inhibit tumor cell growththrough different mechanisms. Resveratrol has been shown to potentiatethe cytotoxic effect of doxorubicin and docetaxel in MCF-7 breast cancercells, and to enhance doxorubicin concentration in other cancer celllines (Al-Abd et al. Cell Prolif 2011; 44(6):591-601). Resveratrol hasalso been shown to enhance cytotoxic effects of vincristine, paclitaxel,and doxorubicin in drug-resistant human epidermoid carcinoma lineKBv200. Resveratrol has also been shown to decrease the expression ofanti-apoptotic bcl-2 and drug efflux pump p-glycoprotein in thesechemoresistant cells (Quan et al. Biomed Pharmacother 2008;62(9):622-629).

Ursolic acid has been shown to decrease viability of a number ofchemoresistant cancer cell types, however the IC₅₀ for UA was stilllower in the parental cells (Shan et al. Chin J Integr Med 2011,17(8):607-611), which have lower levels of p-glycoprotein (Zhang et al.Int J Biochem Cell Biol 2012, 44(8):1244-1253; Shi et al. Eur JPharmacol 2011; 669(1-3):38-44). These results described herein show theeffects of UA can be enhanced by compounds such as resveratrol, whichsubvert traditional resistance phenotypes. The effects of UA andresveratrol were tested in various skin-relevant systems, including invivo mouse skin, human keratinocytes, and skin cancer cell lines todetermine if this combination has a synergistic effect.

IV. FORMULATION AND ADMINISTRATION

Ursolic acid and resveratrol can be administered to a subject eitherorally, parenterally (e.g., intravenously, intramuscularly, orsubcutaneously), intraperitoneally, or locally (for example, powders,ointments or drops). In certain aspects the compounds are provided in anutritional supplement formulation. A nutritional supplement formulationcan be in any form, e.g., liquid, solid, gel, emulsion, powder, tablet,capsule, or gel cap (e.g., soft or hard gel cap). A nutritionalsupplement formulation typically will include one or more compositionsthat have been purified, isolated, or extracted (e.g., from plants) orsynthesized, which are combined to provide a benefit (e.g., a healthbenefit in addition to a nutritional benefit) when used to supplementfood in a diet.

Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions, or emulsions, or may comprise sterile powdersfor reconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents,solvents, or vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), suitable mixtures thereof,triglycerides, including vegetable oils such as olive oil, or injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersions,and/or by the use of surfactants.

These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and/or dispersing agents. Prevention ofmicroorganism contamination of the compositions can be accomplished bythe addition of various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, and the like. Itmay also be desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. Prolonged absorption of injectablepharmaceutical compositions can be brought about by the use of agentscapable of delaying absorption, for example, aluminum monostearateand/or gelatin.

Solid dosage forms for oral administration include capsules, tablets,powders, and granules. In such solid dosage forms, the active compoundis admixed with at least one inert customary excipient (or carrier) suchas sodium citrate or dicalcium phosphate or (a) fillers or extenders, asfor example, starches, lactose, sucrose, mannitol, or silicic acid; (b)binders, as for example, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, or acacia; (c) humectants, as forexample, glycerol; (d) disintegrating agents, as for example, agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certaincomplex silicates, or sodium carbonate; (e) solution retarders, as forexample, paraffin; (f) absorption accelerators, as for example,quaternary ammonium compounds; (g) wetting agents, as for example, cetylalcohol or glycerol monostearate; (h) adsorbents, as for example, kaolinor bentonite; and/or (i) lubricants, as for example, talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, or mixtures thereof. In the case of capsules and tablets, thedosage forms may also comprise buffering agents.

Solid compositions of a similar type may also be used as fillers in softor hard filled gelatin capsules using such excipients as lactose or milksugar, as well as high molecular weight polyethylene glycols, and thelike.

Solid dosage forms such as tablets, capsules, and granules can beprepared with coatings or shells, such as enteric coatings and otherswell known in the art. They may also contain opacifying agents, and canalso be of such composition that they release the active compound orcompounds in a delayed manner. Examples of embedding compositions thatcan be used are polymeric substances and waxes. The active compounds canalso be in micro-encapsulated form, if appropriate, with one or more ofthe above-mentioned excipients.

Liquid dosage forms for oral administration include acceptableemulsions, solutions, suspensions, syrups, and elixirs. In addition tothe active compounds, the liquid dosage form may contain inert diluentscommonly used in the art, such as water or other solvents, solubilizingagents and emulsifiers, as for example, ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide,oils, in particular, cottonseed oil, groundnut oil, corn germ oil, oliveoil, castor oil, sesame seed oil, glycerol, tetrahydrofurfuryl alcohol,polyethylene glycols, fatty acid esters of sorbitan, or mixtures ofthese substances, and the like.

Suspensions, in addition to the active compound(s), may containsuspending agents, as for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol or sorbitan esters, microcrystalline cellulose,aluminum metahydroxide, bentonite, agar-agar, or tragacanth, or mixturesof these substances, and the like.

Dosage forms for topical administration of ursolic acid and resveratrolinclude ointments, powders, sprays and inhalants. The compound(s) areadmixed with a physiologically acceptable carrier, and anypreservatives, buffers, and/or propellants that may be required.

For the compounds of the present invention, alone or as part of asupplement composition, the doses are between about 1, 100, 200, 300,400, 500, 600 to 500, 600, 700, 800, 900, 1000 mg, preferably between200 and 600 mg. In certain aspects, the ratio of ursolic acid toresveratrol can vary between about 4:1, 3:1 2:1, 1:1, 1:2, 1:3 to 1:4.In certain aspects, the compounds are administered 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 times per day. In certain aspects, the compounds areadministered once every 1, 2, 3, 4, 5, 6, or 7 days.

The term “effective amount” means an amount effective, at dosages andfor periods of time necessary, to achieve the desired therapeutic orprophylactic result.

An “effective amount” in reference to decreasing cancer cell growth,means an amount capable of decreasing, to some extent, the growth ofsome cancer or tumor cells. The term includes an amount capable ofinvoking a growth inhibitory, cytostatic and/or cytotoxic effect and/orapoptosis of the cancer or tumor cells. Effective doses will be easilydetermined by one of skill in the art and will depend on the severityand course of the disease, the patient's health and response totreatment, the patient's age, weight, height, sex, previous medicalhistory and the judgment of the treating physician.

The term “subject” means animals, such as dogs, cats, cows, horses,sheep, geese, and humans. Particularly preferred patients are mammals,including humans of both sexes.

The terms “treating”, “treat” and/or “treatment” include preventative(e.g., prophylactic) and palliative treatment.

V. EXAMPLES

The following examples as well as the figures are included todemonstrate embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesor figures represent techniques discovered by the inventors to functionwell in the practice of the invention, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andstill obtain a like or similar result without departing from the spiritand scope of the invention.

A. Example 1—Ursolic Acid and Resveratrol Activate the Insulin and AMPKSignaling Pathways in Skeletal Muscle

Skeletal muscle is the major site for glucose uptake induced by insulin.Insulin binds to the insulin receptor on the muscle cell surface,leading to the activation of several cellular kinases, including Akt andAkt substrate 160 (AS160) facilitating the translocation of the glucosetransporter GLUT 4 to the surface of the muscle cell. Glucose thenenters the muscle cell through GLUT4. In addition, skeletal muscleAMP-activated protein kinase (AMPK) activation promotes glucose uptakeand insulin sensitivity. However, muscle of individuals with type 2diabetes mellitus (T2DM) is resistant to insulin, mainly due to impairedinsulin signaling and glucose transport. Much data show the potentialanti-diabetogenic and anti-inflammatory properties of phytonutrients,such as ursolic acid (UA) found in apples and berries and resveratrol(RES) found in peanuts and grapes.

The purpose of this study was to investigate whether UA activates theinsulin signaling pathway, and whether a combination of UA and RESsynergistically activate AMPK, resulting in GLUT 4 translocation andincreased glucose uptake in muscle cells. L6 myotubes were incubatedwith UA (10 μM) at different time points. Ursolic acid induced Aktphosphorylation by 1.8, 1.7, and 1.8 fold increase at 30, 45, and 60min. Ursolic acid also increased AS160 phosphorylation by 1.3, 2.5, 1.6,and 2.3 fold at 15, 30, 45, and 60 min, respectively. Treatment of L6myotubes with UA (10 μM) or RES (100 μM) for 30 min increased AMPKphosphorylation by 1.7 and 5.3 fold, respectively. A combination of UA(10 μM) and RES (100 μM) for 30 min synergistically induced AMPKphosphorylation by a 7.1 fold increase, that is a 76% increase above UAalone and a 26% increase above RES alone (FIG. 1 ).

Thus, ursolic acid activates the insulin and AMPK signaling pathways inmuscle cells and a combination of ursolic acid and resveratrolsynergistically activate AMPK in muscle cells. This indicates that UAand RES increase the availability of GLUT 4 transporters that can betranslocated to the cell membrane for the uptake of glucose. Since obeseand type 2 diabetic individuals have impaired insulin signaling andglucose uptake, phytonutrients, such as ursolic acid and resveratrol,may represent a novel therapeutic approach to improve insulin action andglucose uptake in these individuals.

Mean Mean [3H]-DPM [3H]-2DG fold corrected for per Min per change CYTO BProtein [ ] from (DPM/mg) SE (pmol/mg/min) SE DMSO Basal 15748.172808.04 185.44 33.06 DMSO 10151.45 2221.79 119.53 26.16 1.00 Ins 100 nM62423.24 11708.83 598.77 29.67 5.01 Met 1 mM 19718.32 4818.90 232.1956.74 1.94 UA 10 uM 21855.37 2248.08 257.35 26.47 2.15 RES 100 uM37819.44 17343.41 244.97 55.90 2.05 UA + RES 34038.17 8303.67 492.6447.44 4.12

B. Example 2—Synergistic Effects of Resveratrol and Ursolic Acid inSkin-Relevant Systems

1. Materials and Methods

Reagents: Ursolic acid (UA), resveratrol (RES), thiazolyl bluetetrazolium bromide (MTT reagent), and an Annexin V/propidium iodidestaining kit were obtained from Sigma (St. Louis, Mo.). For in vivo andkeratinocyte studies, UA was obtained from Santa Cruz Biotechnology(Santa Cruz, Calif.). Human recombinant TNFα was acquired from R&DSystems (Minneapolis, Minn.). 12-O-tetradecanoylphorbol-13 acetate (TPA)was obtained from LC Laboratories (Woburn, Mass.). The CBA MouseTh1/Th2/Th17 Cytokine Kit was obtained from Becton Dickinson (FranklinLakes, N.J.).

Cell culture: HaCaT human keratinocytes were maintained in high glucoseDMEM containing 10% fetal bovine serum, 50 U/ml penicillin, and 50 ng/mlstreptomycin. MT1/2 mouse skin papilloma cells and Ca3/7 mouse skincarcinoma cells were maintained in Joklik MEM containing 8% FBS, 50 U/mlpenicillin, 50 ng/ml streptomycin, 10 μg/ml transferrin, 50 μg/mlgentamicin sulfate, 5 μg/ml insulin, 5 ng/ml EGF, 10 μMo-phosphorylethanolamine, and 10 μM 2-aminoethanol. B16F10 metastaticmouse melanoma cells were grown in DMEM containing 10% FBS, 50 U/mlpenicillin, 50 ng/ml streptomycin. A549 human lung adenocarcinoma cellswere grown in DMEM/F12 containing 10% FBS, 50 U/ml penicillin, and 50ng/ml streptomycin. All cells were grown in an incubator at 5% CO₂ and37° C.

Animals and treatment: Female SENCAR mice, 6-7 weeks old, were purchasedfrom NCI Frederick (Frederick, Md.). The dorsal skin of SENCAR mice wereshaved and treated topically twice per week for 2 weeks with 200 μLacetone vehicle or 1-3 μmol UA, 1-3 μmol RES, or an equimolarcombination of RES and UA. Each treatment was followed 30 minutes laterwith topical application of 200 μL acetone vehicle or 1-2 μg of thetumor promoter TPA.

In vivo sample collection: Animals were sacrificed 6 hr after the finalTPA treatment for Western blotting and 48 hr after the final TPAtreatment for histology. For immunohistochemical (IHC) analysis ofepidermal proliferation five mice per group were i.p. injected with 100mg/kg Bromodeoxyuridine (BrdU) in PBS 30 min prior to sacrifice. 1 cm²of mouse skin from treated areas was fixed in formalin and paraffinembedded to be analyzed via IHC.

Immunohistochemistry (IHC): Slides were immunostained with anti-BrdUantibody. The percentage of BrdU-positive cells in the basal cell layerwas determined from at least 10 randomly selected sites per slide (5mice/group). In addition, the epidermal thickness of each skin samplewas also measured at 10 random sites for 5 mice/group, using ImagePro-Discovery from Media Cybernetics (Bethesda, Md.).

Western blotting: HaCaT cells were treated with sub-optimal doses ofRES, UA, or RES+UA and stimulated with 10 ng/ml human tumor necrosisfactor alpha (TNFα) for 30 min. Cells were scraped into lysis buffercontaining 1% Triton X-100, 0.5% IGEPAL, 0.05 M TrisHCl and 0.1 M NaClas well as protease/phosphatase inhibitors and 5 mM EDT A. For in vivostudies, whole skin was placed on glass over ice and epidermis wasremoved by scraping and placed in RIPA buffer with protease/phosphataseinhibitors. Lysates were homogenized and proteins were extracted bycentrifugation. Twenty micrograms of protein was separated on Bis-Trisgels and transferred onto PVDF membranes. Membranes were blocked in 5%dry milk or BSA and incubated with primary antibodies overnight at 4° C.Membranes were incubated with corresponding HRP-conjugated secondaryantibodies for one hour and developed.

Cytokine analysis: Epidermal samples for each treatment group werepooled and homogenized by mortar and pestle and lysed in 0.05% tween-20in PBS containing 10 mM PMSF. Proteins were extracted by centrifugationand quantified by Bradford. Equivalent protein amounts (100 or 150 μg)per sample were analyzed in triplicate for levels of inflammatorycytokines, including interleukin-2, interleukin-4, interleukin-6,interleukin-10, interleukin-17A, interferon-γ, and tumor necrosisfactor-α using the CBA Mouse Th1/Th2/Th17 Cytokine Kit and an LSR IIflow cytometer (Becton Dickinson) according to manufacturer'sinstructions. Data from each run was normalized to TPA group. Flowcytometry data was generated in the Flow Cytometry Shared ResourceFacility.

Luciferase assay for NFKB activity: HaCaT cells were transfected withthe NFKB Cignal Reporter Assay Kit from Qiagen, using Attractenetransfection reagent, per manufacturer's instructions. Cells were platedat 1.5×10⁴ cells/well in 96 well plates during the transfection. After16 hr transfection, cells were resupplied with OptiMEM containing 0.5%FBS, 1% non-essential amino acids, 50 U/ml penicillin, and 50 ng/mlstreptomycin for 24 hr. Cells were pretreated with various doses of RESor vehicle for 1 hr, followed by UA or vehicle for 1 hr. TNFα at 10ng/ml was added and cells were incubated for 4 hr. Luminescence of boththe TNFα-induced firefly luciferase and the transfection control Renillaluciferase were measured using the Dual-Luciferase Reporter Assay Systemfrom Promega and quantified using a Biotek Synergy HT spectrophotometer.

MTT assay for cell viability: Cells were plated at 1.5×10⁴ cells/well on96-well plates. Cells were pretreated with RES or 0.1% vehicle for 1 hrfollowed by various doses of UA or 0.1% DMSO vehicle for 24 hr. In theexperiment with A549 cells, 5.0×10³ cells/well were plated, and cellswere treated with UA or 0.1% DMSO for 48 hr after 1 hr pretreatment withRES or vehicle. MTT reagent (0.5 mg/ml) was added, and cells wereincubated for an additional 2.5 hr. Media was removed and formazancrystals were dissolved with 100 μl DMSO. Plates were measured at 570 nmwith background subtraction at 650 nm, using a Biotek Synergy HTspectrophotometer.

Annexin V/Propidium Iodide staining for apoptotic/necrotic cells: Ca3/7cells were plated at 2.5×I 05 cells/well on 6-well plates. Cells weretreated with indicated doses of RES, UA, or 0.1% DMSO vehicle for 12 hr.Media and trypsinized cells were collected and spun at 150×g for 3 min,rinsed and spun again, and resuspended in 1× binding buffer containingAnnexin V and propidium iodide according to the manufacturer'sinstructions. Cells were read on an LSR II flow cytometer with deadcells gated out. Flow cytometry data was generated in the Flow CytometryShared Resource Facility.

Statistical Analyses: Differences between individual groups weredetermined by ANOVA. IC₅₀ values were calculated with GraphPad Prism.Resistance Reversal (RR) was calculated by dividing the IC₅₀ value ofthe UA+vehicle group by the IC₅₀ value of the UA+RES group.

2. Results

RES and UA additively prevent TPA-mediated hyperplasia and proliferationin mouse epidermis. In this study, mice were treated topically with thetumor promoter TPA. This study simulates the promotion phase of thetwo-stage chemical carcinogenesis model in mice, in which a chemicaltumor initiator, usually the PAH 7,12-dimethylbenz(a)anthracene (DMBA),and later promoter are applied to the backs of mice. For these studies,the inventors selected doses of UA which had a moderate effect onTPA-induced changes in preliminary studies. Results revealed that RES at1, 2, or 3 μmol total dose had no effect, while the same amounts of UAdose-dependently reversed 2 μg TPA-mediated epidermal thickening. Theeffect of UA was only significantly enhanced by the addition of 2 μmolRES in this study. Also, these doses of RES and UA had limited effect onepidermal proliferation assayed 48 hr after the final TPA treatment.Only 3 μmol of UA alone significantly decreased TPA-induced epidermalproliferation.

The inventors also conducted similar experiments using 1 μg TPA as thepromotion stimulus. The lack of impact of RES and UA on 2 μg TPA-inducedepidermal proliferation may be due to a saturating effect of this highdose. UA dose-dependently decreased the effect of 1 μg TPA on epidermalthickening. In addition, 2 and 3 μmol doses of RES, while having noeffect by themselves, significantly enhanced the anti-hyperplasticeffect of equimolar UA doses. Also, UA dose-dependently decreased 1 μgTPA-induced epidermal proliferation, while 2 μmol RES significantlyenhanced the effect of 2 μmol UA. The combinations of 2 μmol RES+2 μmolUA and 3 μmol RES+3 μmol UA also returned 1 μg TPA-stimulated epidermalproliferation and hyperplasia back to vehicle control levels. Theseresults indicate the potential of RES to enhance UA's anti-tumorpromotion effect.

RES and UA inhibit TPA-mediated inflammatory signaling in mouseepidermis. It was found that RES and UA have an at least additiveinhibitory effect on TPA-mediated NFKB signaling in vivo, measured byphosphorylation of the NFKB subunit p65 and total COX-2 levels. Repeatstudies demonstrated an additive inhibitory effect of RES and UA onTPA-increased p65 phosphorylation and COX-2 levels.

RES and UA synergistically activate AMPK in mouse epidermis. RES and UAhave been shown to activate AMPK, which may contribute to theiranti-inflammatory, anti-diabetic, and/or anti-cancer effects. It wasfound that RES and UA synergistically activate AMPK in mouse epidermis(FIG. 2 ). Repeat experiments also showed the combination of RES and UAactivated AMPK in the epidermis. Activation of AMPK may contribute tothe decrease in epidermal proliferation, hyperplasia, and inflammatorysignaling mediated by the combination of RES and UA. In addition, theseresults indicate the combination of RES and UA may also synergisticallyameliorate diseases inversely associated with AMPK signaling, such asinsulin resistance.

RES and UA additively inhibit TNFα-mediated NFKB activity in humankeratinocytes. As mentioned before, NFKB activity is important duringthe tumor promotion phase. The inventors conducted studies in HaCaThuman keratinocytes to determine if RES and UA could additively orsynergistically inhibit NFKB activity. Cells were pretreated with dosesof RES (100 and 200 μM) and UA (10 and 20 μM), which had a moderateeffect on TNFα-induced NFKB activity in preliminary experiments(measured by phosphorylation of active subunit p65). Combinations of RESand UA generally had an additive inhibitory effect on TNFα-mediated p65phosphorylation.

Downstream NFKB transcriptional activity was also analyzed vialuciferase reporter gene assay. For combination experiments, UA doseswhich had a moderate effect on TNFα-induced NFKB activity were selected.UA dose-dependently decreases TNFα-stimulated NFKB activity, and thiseffect is at least partially reversed with 100 μM RES. In anotherexperiment, increasing doses of RES were combined with 20 μM UA. Athigher doses of RES (100 and 200 μM), which partially inhibited NFKBthemselves, the combination of RES and UA has an additive effect. Theinventors observed an antagonistic effect of lower doses of RES (50 μM)with UA.

RES enhances the cytotoxic effects of UA in skin cancer cell lines. Inboth MT1/2 mouse papilloma cells and Ca3/7 mouse squamous carcinomacells, increasing doses of RES lowered the IC₅₀ values of UA. The foldby which the IC₅₀ is decreased is indicated by resistance reversal(FIGS. 3A-B), and is dose dependently enhanced by RES. In another study,the ability of 200 μM RES to enhance UA-mediated growth suppression wastested. RES strongly and significantly synergized with UA (FIG. 4 ). Thesynergistic effects of RES and UA in the Ca3/7 cell line were confirmedby flow cytometry, which showed an enhancement of mixedapoptotic/necrotic cell death. In addition, the synergistic effects ofRES and UA were also evident in the highly metastatic mouse melanomacell line B16F10 as well as in the human lung adenocarcinoma cell lineA549 (FIG. 5 ).

The invention claimed is:
 1. A method of decreasing obesity in a human,the method comprising: eating, one or more times per day, an effectiveamount of a nutritional supplement comprising ursolic acid andresveratrol in a ratio of ursolic acid to resveratrol of 3:1 to 1:3 todecrease obesity in the human; continuing eating an effective amount ofthe nutritional supplement for multiple days; and decreasing obesity inthe human; wherein the nutritional supplement is formulated as a tablet,a capsule, a concentrate, a powder, a drink, or a food, and is eaten inaddition to food in a normal diet.
 2. The method of claim 1, wherein theeffective amount of the nutritional supplement comprises 50 to 600 mg ofursolic acid.
 3. The method of claim 1, wherein the effective amount ofthe nutritional supplement comprises 200 mg of ursolic acid.
 4. Themethod of claim 1, wherein the effective amount of the nutritionalsupplement comprises 50 to 600 mg resveratrol.
 5. The method of claim 1,wherein the effective amount of the nutritional supplement comprises 600mg of resveratrol.
 6. The method of claim 1, wherein the effectiveamount of the nutritional supplement comprises 200 mg of ursolic acidand 600 mg of resveratrol.
 7. The method of claim 1, wherein theeffective amount of the nutritional supplement comprises 100 mg ofursolic acid and 200 mg of resveratrol.
 8. The method of claim 1,wherein the nutritional supplement comprises one or more of apreserving, an emulsifying, or a dispersing agent.
 9. The method ofclaim 1, wherein the nutritional supplement comprises one or more of afiller or extender, a binder, a humectant, a disintegrating agent,solution retarder, absorption accelerator, wetting agent, an adsorbent,lubricant, or buffering agent.
 10. The method of claim 9, wherein thefiller is starch, lactose, sucrose, mannitol, or silicic acid.
 11. Themethod of claim 9, wherein the binder is carboxymethylcellulose,alginates, gelatin, polyvinylpyrrolidone, sucrose, or acacia.
 12. Themethod of claim 9, wherein the humectant is glycerol.
 13. The method ofclaim 9, wherein the disintegrating agent is agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, complex silicates, orsodium carbonate.
 14. The method of claim 9, wherein the solutionretarder is paraffin.
 15. The method of claim 9 wherein the wettingagent is cetyl alcohol or glycerol monostearate.
 16. The method of claim9, wherein the lubricant is talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof.17. The method of claim 9, wherein the nutritional supplement is a bakedgood, chocolate, caramel, cookie, or bar.
 18. The method of claim 1,wherein the nutritional supplement is a baked good, chocolate, caramel,cookie, or bar.